Ultrastructural characteristics of human granulosa cells in a coculture system for in vitro fertilization

Abstract

The use of somatic cells for cocultures during in vitro fertilization (IVF) is currently finalized to obtain a higher number of healthy and viable embryos with a high potential of implantation. Among the different cell lines that can be used as feeder cells for cocultures, granulosa cells (GCs) are autologous cells, safe and easy to recover. The aim of the present study was to analyze the fine structure of human GCs used in a coculture system to evaluate, from a morphodynamic point of view, their role in supporting embryo development. GCs were collected during oocyte pick-up, 36 h after human chorionic gonadotropin administration, from patients undergoing IVF procedures, who had given their informed consent to be included in this protocol. After coculture, GCs were fixed and processed for light microscopy (LM) and transmission electron microscopy (TEM). By LM, GCs appeared as clusters of loosely packed cells, irregularly rounded or polyhedral in shape, varying in diameter from 18 to 25 microm. Mitotic cells, as well as regressing elements (with pyknotic nuclei or dense cytoplasm) and cell fragments were occasionally observed. By TEM, the plasma membrane was irregular due to the presence of cytoplasmic evaginations. Linear and annular gap junctions between neighboring GCs were found. GC nuclei, rounded and eccentrically located, contained finely dispersed chromatin, one (often two) prominent nucleoli and, infrequently, peripheral patches of heterochromatin. Numerous organelles populated the GC cytoplasm, among them, mitochondria were rod-shaped or elongated, usually provided with tubular-vesicular cristae but occasionally showing atypical, longitudinally oriented cristae. Membranes of smooth endoplasmic reticulum, Golgi stacks and vesicles, secretory-like granules, cisternae of rough endoplasmic reticulum (RER), free ribosomes and polysomes, lysosomal-like bodies, microfilaments, and lipid droplets were also seen in the GC cytoplasm. In most cells, RER was scarcely represented and numerous lipid droplets filled the perinuclear space. On the contrary, some GCs contained an abundant RER and rare lipid droplets scattered in the cytoplasm. In conclusion, our data demonstrated the presence, in a coculture system, of GCs provided with ultrastructural characteristics typical of healthy, metabolically active, mostly steroidogenic cells. Protein-synthetic cells have also been detected. These data, evaluated at the light of biochemical and clinical studies, sustain the capability of human GCs cocultures to positively affect early embryo development in vitro by the secretion of steroids and proteins, putative "embryotrophic" factors.

Leptin-deficient (ob/ob) mouse ovaries show fatty degeneration, enhanced apoptosis and decreased expression of steroidogenic acute regulatory enzyme

“…Annular junctions arise from phagocytised gap junctions as described for granulosa cells from human preovulatory follicles. 37,38 We here interpret the frequent observation of annular junctions in context with the increased follicular atresia.…”

Thyroid hormones induce cell proliferation and survival in ovarian granulosa cells COV434

“…Other authors have similarly shown granulosa cells with abundant RER (Nottola et al, 2006) and have considered them as G-rich protein-synthetic cells. However, it has also been demonstrated that, in cell culture, the numerous polyribosomes and, in some circumstances, a disaggregation of polyribosomes are connected to a marked depression of protein synthesis during mitosis (Ghadially, 1988).…”

Differences in developmental competence and gene expression profiles between buffalo (Bubalus bubalis) preimplantation embryos cultured in three different embryo culture media

“…In nearly all studies on buffalo IVEP, presumptive zygotes are subject to an in vitro culture (IVC) system, involving use of TCM-199, a complex medium coculture with buffalo oviductal epithelial cells or granulosa cells and supplemented with serum, even though it has been demonstrated that primary oviduct (Mugnie et al 2009) and granulosa cell (Nottola et al 2006) cultures secrete a series of factors that act in a paracrine manner stimulating development to the blastocyst stage. This system is complex and associated blastocyst yield is very low.…”